Compositions and methods for the inhibition of membrane fusion by paramyxoviruses

ABSTRACT

Fusion of the membrane of enveloped viruses with the plasma membrane of a receptive host cell is a prerequisite for viral entry and infection and an essential step in the life cycle of all enveloped viruses, such as paramyxoviruses. The instant invention is directed to providing polypeptides which are a heptad portion of a Henipavirus F protein effective against fusion between a membrane of a paramyxovirus and a plasma membrane of a cell. The instant invention also provides nucleic acids, compositions, and methods effective against paramyxovirus infection. Accordingly, the instant invention provides therapeutic agents and vaccines effective against paramyxoviruses viruses, especially HeV or NiV.

REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.60/331,231 of the same title filed Nov. 13, 2001, the entire disclosureof which is incorporated by reference in its entirety.

RIGHTS IN THE INVENTION

This invention was made, in part, with support from the United StatesGovernment, grant no. RO731L. Accordingly, the United States Governmentmay have certain rights to this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to peptides, compositions and methods involvingthese peptides for the inhibition of membrane fusion by paramyxovirusesand, in particular, membrane fusion mediated by Hendra virus and Nipahvirus.

2. Description of the Background

Membrane fusion is a ubiquitous cell biological process. Fusion eventswhich mediate cellular housekeeping functions, such as endocytosis,constitutive secretion, and recycling of membrane components, occurcontinuously in all eukaryotic cells.

Additional fusion events occur in specialized cells. Intracellularly,for example, fusion events are involved in such processes as occur inregulated exocytosis of hormones, enzymes and neurotransmitters.Intercellularly, such fusion events feature prominently in, for example,sperm-egg fusion and myoblast fusion.

Fusion events are also associated with disease states. For example,fusion events are involved in the formation of giant cells duringinflammatory reactions, and particularly, the entry of all envelopedviruses into cells.

For example, the paramyxoviruses are negative-stranded RNA containingenveloped viruses encompassing a variety of important human and animalpathogens including measles virus (MeV), canine distemper virus (CDV),human parainfluenza viruses (hPIV) 1-4, and simian virus 5 (SV5)(reviewed in (Lamb and Kolakofsky, 1996)). These viruses contain twoprincipal membrane-anchored glycoproteins which appear as spikesprojecting from the envelope membrane of the viral particle when imagedin the electron microscope. One glycoprotein is associated with virionattachment to the host cell and, depending on the particularparamyxovirus, has been designated as either thehemagglutinin-neuraminidase protein (HN), the hemagglutinin protein (H),or the G protein which has neither hemagglutinating nor neuraminidaseactivities. The attachment glycoproteins of the paramyxoviruses are typeII integral membrane proteins. In several cases where the attachmentglycoprotein is of the HN type, it is sialic acid moieties which serveas receptors for virus entry. For paramyxoviruses possessing an H or Gattachment glycoprotein the identity of host cell receptors are notknown, except in the case of MeV where CD46 can serve as a functionalreceptor (Naniche et al., 1993). The second glycoprotein is the fusionprotein (F), a type I membrane glycoprotein, which facilitates themembrane fusion event between the virion and host cell during virusinfection (reviewed in Lamb, 1993).

Fusion of the membrane of enveloped viruses with the plasma membrane ofa receptive host cell is a prerequisite for viral entry and infectionand an essential step in the life cycle of all enveloped viruses.Following paramyxovirus attachment to a permissive host cell, fusion atneutral pH between the virion and plasma membranes ensues, resulting indelivery of the nucleocapsid into the cytoplasm (reviewed in (Lamb andKolakofsky, 1996)). In a related process, cells expressing these viralglycoproteins at their surfaces can fuse with receptor-bearing cells,resulting in the formation of multinucleated giant cells (syncytia). Theparamyxovirus F glycoprotein shares several features with other viralmembrane fusion proteins, including the envelope glycoprotein ofretroviruses like gp120/gp41 of HIV-1, and hemagglutinin (HA) ofinfluenza virus (reviewed in (Hernandez et al., 1996)). The biologicallyactive F protein consists of two disulfide linked subunits, F₁ and F₂,that are generated by the proteolytic cleavage of a precursorpolypeptide known as F₀ (reviewed in (Klenk and Garten, 1994; Scheid andChoppin, 1974)). In all cases the membrane-anchored subunit contains anew amino terminus that is hydrophobic and highly conserved across virusfamilies and referred to as the fusion peptide (reviewed in (Hunter,1997)). The fusion peptide is an important structural element requiredto mediate virion/host cell membrane fusion. All paramyxoviruses studiedto date, with the exception of SV5, under certain circumstances, requireboth the attachment and F glycoprotein for membrane fusion (Paterson,Johnson, and Lamb, 1997). Although evidence of physical interactionshave only been rarely detected, it is hypothesized that the bindingprotein must somehow signal and induce a conformational change in Fleading to virion/host cell membrane fusion (Lamb, 1993).

In 1994, a new paramyxovirus, now called Hendra virus (HeV) andrecognized to be a member of the subfamily Paramyxovirinae, was isolatedfrom fatal cases of respiratory disease in horses and humans, and wasshown to be distantly related to MeV and other members of themorbillivirus genus (Murray et al., 1995). The first outbreak of severerespiratory disease in the Brisbane suburb of Hendra resulted in thedeath of 13 horses and their trainer, and the non-fatal infection of astable hand and a further 7 horses. At approximately the same time, inan unrelated incident almost 100 km north of Hendra, a 35-year-old manexperienced a brief aseptic meningitic illness after caring for andassisting at the necropsies of two horses subsequently shown to havedied as a result of HeV infection. Thirteen months later the mansuffered severe encephalitis characterized by uncontrolled focal andgeneralized epileptic-activity. A variety of studies that were performedin the evaluation of this fatality, including serology, PCR, electronmicroscopy and immunohistochemistry, strongly suggested that HeV wasindeed the cause of this patient's encephalitis, and the virus wasacquired from the HeV-infected horses (O'Sullivan et al., 1997). In all,fifteen horses and two people died in the two episodes. At the time thesource of the emerging virus was undetermined, but more recently it hasbeen found that approximately 50% of Australian fruit bats, commonlyknown as flying foxes, have antibodies to HeV and HeV-like viruses havebeen isolated from bat uterine fluids and it appears that these animalsare the natural host for the virus (Field et al., 2001; Halpin et al.,1999; Halpin et al., 2000; Young et al., 1996).

More recently, the nucleic acid sequence of the genes of HeV has beencompared with those of other paramyxoviruses (Wang et al., 1998; Yu etal., 1998a; Yu et al., 1998b). These later studies have confirmed thatHeV is a member of the Paramyxoviridae, subfamily Paramyxovirinae, butlow homology with other subfamily members and the presence of severalnovel biological and molecular features such as F protein cleavage at asingle lysine residue and genome length suggest classification in a newgenus within the Paramyxovirinae.

Subsequent to these events, an outbreak of severe encephalitis in peoplewith close contact exposure to pigs in Malaysia and Singapore occurredin 1998 (Anonymous, 1999). The outbreak was first noted in lateSeptember 1998 and by mid-June 1999, more than 265 cases ofencephalitis, including 105 deaths, had been reported in Malaysia, and11 cases of encephalitis or respiratory illness with one death had beenreported in Singapore. This may represent a near 40% fatality rate uponinfection, because the incidence of subclinical human infections duringthese episodes has not been well defined. Electron microscopic,serologic, and genetic studies have since indicated that this virus alsobelongs to the Paramyxovirinae subfamily, and was most closely relatedto HeV. This virus was named Nipah virus (NiV) after the small town inMalaysia from which the first isolate was obtained from thecerebrospinal fluid of a fatal human case (Chua et al., 2000; Chua etal., 1999; Goh et al., 2000; Lee et al., 1999; Lim et al., 2000). NiVand HeV are now recognized as the prototypic members of a new genuswithin the Paramyxovirinae subfamily called Henipavirus (Wang et al.,2001; Wang and Eaton, 2001). Both HeV and NiV are unusual among theparamyxoviruses in their ability to infect and cause potentially fataldisease in a number of host species, including humans and in that theyhave an exceptionally large genome.

These viruses classified as Biosafety Level 4 agents (BSL-4) and havethe potential to be used as biological warfare agents. There are noexisting antiviral therapies effective against these viruses, and theonly therapies in existence to any viruses in the paramyxovirus familyare attenuated vaccines for the prevention of infection by MeV and Mumpsvirus. Accordingly, agents and compositions effective against infectionsby viruses in the paramyxovirus family are particularly desirable. Inthis connection, the heptad peptides of the instant invention representan effective therapy for paramyxovirus infection resulting from, e.g., abiological weapon, a natural outbreak, or a BSL-4 laboratory accident.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides effectivetreatments against infection by a paramyxovirus of the subfamilyHenipavirus.

One embodiment of the invention is directed to peptides and compositionscontaining peptides that are useful for treating infection by viruses ofthe subfamily Henipavirus by inhibiting membrane fusion induced by HeVand NiV. Such peptides comprise the amino acid sequence of the Fglycoprotein of Henipaviruses and preferably the heptad peptide of theC-terminal region. Most preferably the peptide is SEQ ID NO 1 or SEQ IDNO 2. This embodiment further comprises nucleic acids the encodepeptides of the invention.

Another embodiment of the invention is directed to methods for thetreatment of infection by viruses of the subfamily Henipavirus byadministering peptides of the invention to a patient.

Another embodiment of the invention is directed to antibodies topeptides of the invention and diagnostics containing these antibodies.

In another embodiment, the invention provides a recombinant or isolatedpolypeptide comprising a heptad portion of a Henipavirus F protein andbiologically active fragments thereof and variants thereof.

Preferably, the polypeptide comprises the polypeptide sequence ofselected from the group consisting of SEQ ID NO 1, SEQ ID NO 2,biologically active fragments thereof, and variants thereof. Preferably,the polypeptide functions to inhibit fusion of a membrane of aparamyxovirus and a plasma membrane of a cell. Preferably, the heptadportion is derived from HeV or NiV.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising an effective amount of a polypeptide sequence ofselected from the group consisting of SEQ ID NO 1, SEQ ID NO 2,biologically active fragments thereof, and variants thereof, and apharmaceutically acceptable carrier. The composition of claim 5, whereinthe composition is a therapeutic or post-exposure prophylactic.Preferably, the composition is in the form of a vaccine.

In another embodiment, the invention provides a method for inhibitingfusion between a membrane of a paramyxovirus and a plasma membrane of acell comprising administering a composition according to the instantPreferably, the paramyxovirus is of the genus Henipavirus. Preferably,the paramyxovirus is of the subfamily Paramyxovirina. Preferably,paramyxovirus is HeV or NiV.

In another embodiment, the invention provides an isolated polynucleotidesequence encoding a polypeptide that inhibits fusion between a membraneof a paramyxovirus and a plasma membrane of a cell, wherein saidpolynucleotide is either a DNA sequence encoding a polypeptide of SEQ IDNO 1; and a DNA sequence capable of hybridizing under high stringencyconditions to the complement of (a).

In another embodiment, the invention provides a vector comprising apolynucleotide sequence of the instant invention.

In another embodiment, the invention provides a cell comprising apolynucleotide sequence of the instant invention.

In another embodiment, the invention provides a method for treatinginfection with a virus, comprising administering a composition accordingthe instant invention.

In another preferred embodiment. The instant invention provides aptamersof peptides of the instant invention and methods of using the same.

Other features and advantages of the invention will become apparent fromthe following description. It should be understood, however, that thedescription and the examples are given for illustration, and variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those skilled in the art from this description.

DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate a presently preferred embodimentof the invention, and, together with the general description given aboveand the detailed description of the preferred embodiment providedherein, serve to explain the principles of the invention. Thus, for amore complete understanding of the present invention, the objects andadvantages thereof, reference is now made to the following descriptionstaken in connection with the accompanying drawings in which:

FIG. 1 (A) Representation of the HeV F glycoprotein depicting importantstructural and functional elements. (B) Amino acid sequence of theC-terminal heptad repeat of HeV F (SEQ ID NO 1). (C) Helical wheelrepresentation of the N- and C-terminal heptad repeats of HeV F.

FIG. 2 Graphs of β-gal activity verses increasing peptide concentration.A: Inhibition of HeV-mediated fusion by C-terminal synthetic F peptides.B: Inhibition of NiV-mediated fusion by C-terminal synthetic F peptides.

FIG. 3 A comparison of peptide concentration of FC1 or FC 2 on thenumber of nuclei in NiV infected cells.

DESCRIPTION OF THE INVENTION

As embodied and broadly described herein, the present invention isdirected to effective treatments against infection by a paramyxovirus.More specifically, the present invention relates to peptides that areuseful at inhibiting membrane fusion induced by viruses of the subfamilyHenipavirus such as, for example, HeV and NiV.

Understanding the mechanisms of how paramyxoviruses emerge, mediate hostcell infection and cross species, is an important step towardsdetermining how to address such new, emerging, and sometimes reemerginginfectious disease threats. To this end, examination of HeV was begunwith the characterization and use of the virus' envelope glycoproteinsthat facilitate attachment and membrane fusion events during infection.HeV possesses a F glycoprotein, similar to other paramyxoviruses, thatlikely mediates membrane fusion. The attachment glycoprotein has beendesignated G, like that of respiratory syncytial virus, because, on thebasis of genetic analyses and observations with infectious virus, HeVcontains neither hemagglutinating nor neuraminidase activity (Murray etal., 1995), suggesting that the cellular receptor may not be sialic acid(Yu et al., 1998a).

All viral membrane glycoproteins that are the mediators of membranefusion, virion attachment or both, are invariably oligomeric (Doms etal., 1993). Considerable advances in the understanding of the structuralfeatures of these oligomeric viral envelope glycoproteins has beenattained in recent years and have centered on the influenza virus andHuman Immunodeficiency virus (HIV) systems. A notable structural featureof many of these fusion glycoproteins is the presence of 2 α-helicaldomains referred to as heptad repeats that are important for botholigomerization and function of the glycoprotein, where they areinvolved in the formation of a trimer-of-hairpins structure (Hughson,1997; Singh, Berger, and Kim, 1999). Peptides corresponding to either ofthese domains can potently inhibit the fusion process, first noted withsequences derived from the gp41 subunit of HIV-1 envelope glycoprotein(Jiang et al., 1993; Wild et al., 1994). Inhibition of the formation ofthe trimer-of-hairpins structure inhibits the fusion process, and thismechanism has been modeled and described by several groups (Chan andKim, 1998; Munoz-Barroso et al., 1998; Rimsky, Shugars, and Matthews,1998; Root, Kay, and Kim, 2001). Indeed, the development and clinicalapplication of fusion-inhibitors, as antiviral therapies for HIV-1, hasbeen a direct result from this area of research. Recently, an α-helicaltrimeric core complex has been defined in the F protein of Simian virus5 (SV5) and is also believed to be either the fusion competent structureor the structure formed after fusion has occurred, analogous to HIV-1gp41 (Lamb, Joshi, and Dutch, 1999). In addition, peptide sequences fromthe C-terminal heptad of SV5 F, as well as Measles virus (MeV) F, havebeen shown to be potent inhibitors of membrane fusion (Joshi, Dutch, andLamb, 1998; Wild and Buckland, 1997).

Heptad repeats of Hendra virus (HeV) and Nipah virus (NiV) F wereanalyzed using helical wheel diagrams and sequences were identified thatmay inhibit virus-mediated fusion (FIG. 1). Next, the specificity ofrecombinant HeV fusion system was tested using a synthetic 42 amino acidpeptide (FC 1) corresponding to the HeV F C-terminal heptad. It wassurprisingly discovered that the FC1 peptide could completely inhibitHeV-mediated fusion in the nM range (FIG. 2). It is believed thispeptide will also inhibit live HeV infection when tested under BSL-4conditions, and represents a therapeutic avenue for both HeV and NiVinfections. Indeed, the HeV F C-terminal heptad peptide was also capableof inhibiting recombinant NiV-mediated fusion at slightly lowerefficiencies likely due to several mismatches in the heptad sequence.The exact NiV C-terminal heptad peptide is being synthesized and tested.The use of such peptide inhibitors to HeV and NiV may be the onlyeffective therapy during acute encephalitic episodes caused by infectionwith these agents in either humans or livestock such as valuable racehorses or polo ponies.

In one embodiment, the invention is directed to compositions containingall or effective portions of a peptide corresponding to the F protein ofHenipavirus. Preferably, the peptide is the C-terminal heptad peptide ofHeV, which has been localized to position 442 to 489 of the Fglycoprotein. This peptide has been shown to significantly reducemembrane fusion of HeLa cells infected with recombinant vaccinia viruscontaining HeV F and G proteins. Other viruses of the subfamilyHenipavirus, including those yet to be discovered, that have acorresponding region in their F protein, such as, for example, NiV Fprotein, are also effective at inhibiting membrane fusion and as atreatment against infection. Compositions are effective both in vitroand also in vivo in the treatment and prevention of infection byHenipavirus in cells or in mammals such as, for example, horses, sheep,pigs, cattle and humans. Compositions may contain only peptide of theinvention or additional ingredients such as pharmaceutically acceptableagents including carbohydrates, saccharides, polysaccharides, lipids,fatty acids, proteins or protein fragments, glycol, polyethylene glycol,glycerol, glucose, oil, water, cellulose, and other similar substances.Preferably, peptides are physiologically stable in the cells or patientfor an time sufficient for the effective inhibition of membrane fusion.

The instant proteins, peptides and polypeptides describe any chain ofamino acids, regardless of length or post-translational modification(for example, glycosylation or phosphorylation) and apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid.Thus, the instant polypeptides include full-length, naturally occurringproteins as well as recombinantly or synthetically produced polypeptidesthat correspond to a full-length naturally occurring protein or toparticular domains or portions of a naturally occurring protein. Thepolypeptides also encompass mature proteins which have an addedamino-terminal methionine to facilitate expression in prokaryotic cells.

The invention includes biologically active polypeptides and biologicallyactive fragments thereof which refers to the ability to inhibit fusionbetween a membrane of a paramyxovirus and a plasma membrane of a hostcell.

The present invention contemplates modification of the instantpolypeptides to create variants. Such modifications may be deliberate,as by site-directed mutagenesis, or may be spontaneous. All of thepolypeptides produced by these modifications are included herein as longas the ability to inhibit fusion between a membrane of a paramyxovirusand a plasma membrane of a host cell is present.

For example, the present invention contemplates the deletion of one ormore amino acids from the instant polypeptides to create deletionvariants. This deletion can be of one or more amino or carboxy terminalamino acids or one or more internal amino acids (e.g. silentsubstitutions or mutations). The present invention further contemplatesone or more amino acid substitutions to the instant polypeptides tocreate subsititutional variants. The present invention contemplates thatsuch substitutional variants would contain certain functionalalterations, such as stabilizing against proteolytic cleavage. Yet, itis understood that such variants retain their inhibitory activity.

Substitutions preferably are conservative, that is, one amino acid isreplaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparigine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparigine; glutamate to aspartate; glycine to proline;histidine to asparigine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine, glutamine, orglutamate; methionine to leucine or isoleucine; phenylalanine totyrosine, leucine or methionine; serine to threonine; threonine toserine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine;and valine to isoleucine or leucine. Other substitutions are routine orwell known in the art and are well within the scope of the instantinvention.

The present invention further contemplates the insertion of one or moreamino acids to the instant polypeptides sequences to create aninsertional variant. Examples of such insertional variants includefusion proteins such as those used to allow rapid purification of thepolypeptide and also can include hybrid polypeptides containingsequences from other proteins and polypeptides which are homologues ofthe inventive polypeptide. For example, an insertional variant couldinclude portions of the amino acid sequence of the polypeptide from onespecies, together with portions of the homologous polypeptide fromanother species. Other insertional variants can include those in whichadditional amino acids are introduced within the coding sequence of thepolypeptides. These typically are smaller insertions than the fusionproteins described above and are introduced, for example, to disrupt aprotease cleavage site.

Polypeptides of the present invention can be synthesized by suchcommonly used methods as t-BOC or FMOC protection of alpha-amino groups.Both methods involve step-wise syntheses whereby a single amino acid isadded at each step starting from the C terminus of the peptide (Coliganet al., Current Protocols in Immunology, Wiley Interscience, Unit 9,1991). In addition, polypeptide of the present invention can also besynthesized by solid phase synthesis methods (e.g., Merrifield, J. Am.Chem. Soc. 85:2149, 1962; and Steward and Young, Solid Phase PeptideSynthesis, Freeman, San Francisco pp. 27-62, 1969) using copolyol(styrene-divinylbenzene) containing 0.1-1.0 mM amines/g polymer. Oncompletion of chemical synthesis, the polypeptides can be deprotectedand cleaved from the polymer by treatment with liquid HF 10% anisole forabout 15-60 min at 0° C. After evaporation of the reagents, the peptidesare extracted from the polymer with 1% acetic acid solution, which isthen lyophilized to yield crude material. This can normally be purifiedby such techniques as gel filtration of Sephadex G-15 using 5% aceticacid as a solvent. Lyophilization of appropriate fractions of the columnwill yield a homogeneous polypeptide or polypeptide derivatives, whichare characterized by such standard techniques as amino acid analysis,thin layer chromatography, high performance liquid chromatography,ultraviolet absorption spectroscopsy, molar rotation, solubility andquantitated by solid phase Edman degradation.

The instant polypeptides include their pharmacologically acceptablesalts by treatment with physiologically acceptable acids or bases. Thesesalts include, for example, salts with an inorganic acid such ashydrochloric acid, sulfuric acid and nitric acid and, depending on thecompounds, salts with an organic acid such as acetic acid, nitric acid,succinic acid and maleic acid, salts with an alkali metal such as sodiumand potassium, and salts with an alkaline earth metal such as calcium.The instant polypeptides also include all their stereoisomeric formsincluding their diastereomers.

In a preferred embodiment, the invention is directed to nucleic acidsthat encode peptides of the invention. These may comprise DNA, RNA orPNA and contain sequences in addition to sequences that encode thepeptide. Such additional sequences may include transcription ortranslation controlling sequences such as promoter or operatorsequences, transcription initiation sites, splice junctions, terminationsites, origins of replication and other control sequences. Preferably,the nucleic acid is an expression vector that allows for the controlledexpression (e.g. inducible expression) of the encoded peptide. Suitablevectors include eukaryotic vectors such as viral vectors, prokaryoticvectors such as plasmids, and shuttle vectors.

The instant polynucleotide is a polymer of deoxyribonucleotides orribonucleotides in the form of a separate fragment or as a component ofa larger construct In a preferred embodiment, the invention provides aDNA sequence encoding a polypeptide of SEQ ID NO 1 or SEQ ID NO 2; andDNA sequence capable of hybridizing under high stringency conditions tothe complement of a DNA sequence encoding a polypeptide of SEQ ID NO 1or SEQ ID NO 2.

Polynucleotide sequences of the invention include DNA, RNA and cDNAsequences. Preferably, the polynucleotide sequences encoding the instantpolypetides is the sequence of SEQ ID NO 1 or SEQ ID NO 2. DNA sequencesof the present invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare known in the art. Such hybridization procedures include, forexample, hybridization of probes to cDNA libraries to detect sharednucleotide sequences, antibody screening of expression libraries todetect shared structural features, such as a common antigenic epitope,and synthesis by the polymerase chain reaction (PCR), or syntheticchemical synthesis of the instant polypeptide sequence.

Hybridization procedures are useful for screening of recombinant clonesby using labeled mixed synthetic oligonucleotide probes, wherein eachprobe is potentially the complete complement of a specific DNA sequencein a hybridization sample which includes a heterogeneous mixture ofdenatured double-stranded DNA. For such screening, hybridization ispreferably performed on either single-stranded DNA or denatureddouble-stranded DNA. Hybridization is particularly useful for detectionof cDNA clones derived from sources where an extremely low amount ofmRNA sequences relating to the polypeptide of interest are present.Using stringent hybridization conditions directed to avoid non-specificbinding, it is possible to allow an autoradiographic visualization of aspecific cDNA clone by the hybridization of the target DNA to thatsingle probe in the mixture, which is its complement (Wallace et al.Nucl. Acid Res. 9:879, 1981). Stringent conditions preferably includehigh stringency conditions. See, for example, Maniatis et al, MolecularCloning (A Laboratory Manual), Cold Spring Harbor Laboratory, pages387-389, 1982. One such high stringency hybridization condition is, forexample, 4×SSC at 65° C., followed by washing in 0.1×SSC at 65° C. forthirty minutes. Alternatively, another high stringency hybridizationcondition is in 50% formamide, 4×SSC at 42° C.

The development of specific DNA sequences encoding the instantpolypeptides can also be obtained by isolation of double-stranded DNAsequences from the genomic DNA, chemical manufacture of a DNA sequenceto provide the necessary codons for the polypeptide of interest, and invitro synthesis of a double-stranded DNA sequence by reversetranscription of mRNA isolated for a eukaryotic donor cell. In thelatter case, a double-stranded DNA complement of mRNA is eventuallyformed which is generally referred to as cDNA.

The synthesis of DNA sequences is frequently a method that is preferredwhen the entire sequence of amino acids residues of the desiredpolypeptide product is known. When the entire sequence of amino acidresidues of the desired polypeptide is not known, direct synthesis ofDNA sequences is not possible and it is desirable to synthesize cDNAsequences. cDNA sequence isolation can be done, for example, byformation of plasmid- or phage-carrying cDNA libraries which are derivedfrom reverse transcription of mRNA. mRNA is abundant in donor cells thathave high levels of genetic expression. In the event of lower levels ofexpression, PCR techniques are preferred. When a significant portion ofthe amino acid sequence is known, production of labeled single or doublestranded DNA or RNA probe sequences duplicating a sequence putativelypresent in the target cDNA may be employed in DNA/DNA hybridizationprocedures, carried out on cloned copies of the cDNA (denatured into asingle-stranded form) (Jay et al., Nucl. Acid Res. 11:2325, 1983).

The polynucleotides of this invention include sequences that aredegenerate as a result of the genetic code. The genetic code isdescribed as degenerate because more than one nucleotide triplet, calleda codon, can code for a single amino acid. The present inventioncontemplates the degeneracy of the genetic code and includes alldegenerate nucleotide sequences which encode.

The present invention further includes allelic variations, i.e.,naturally-occurring base changes in a species population which may ormay not result in an amino acid change, to the polynucleotide sequencesencoding a polypeptide of SEQ ID NO 1 or SEQ ID NO 2; and DNA sequencecapable of hybridizing under high stringency conditions to thecomplement of a DNA sequence encoding a polypeptide of SEQ ID NO 1 orSEQ ID NO 2. The inventive polynucleotide sequences further comprisethose sequences which hybridize under high stringency conditions (see,for example, Maniatis et al, Molecular Cloning (A Laboratory Manual),Cold Spring Harbor Laboratory, pages 387-389, 1982) to the codingregions or to the complement of the coding regions of a polypeptide ofSEQ ID NO 1. One such high stringency hybridization condition is, forexample, 4×SSC at 65° C., followed by washing in 0.1×SSC at 65° C. forthirty minutes. Alternatively, another high stringency hybridizationcondition is in 50% formamide, 4×SSC at 42° C.

The instant vector is a DNA molecule, such as a plasmid, cosmid, orbacteriophage that has the capability of replicating autonomously in ahost cell. Cloning vectors may preferably contain (i) one or a smallnumber of restriction endonuclease recognition sites at which foreignDNA sequences can be inserted in a determinable fashion without loss ofan essential biological function of the vector, and (ii) a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes may typically includegenes that provide tetracycline resistance or ampicillin resistance.

The cell is a naturally occurring cell or a transformed cell thatcontains an expression vector and supports the replication or expressionof the expression vector. Host cells may be cultured cells, explants,cells in vivo, and the like. Host cells may be prokaryotic cells such asE. coli, or eukaryotic cells such as yeast, insect, amphibian, ormammalian cells such as CHO, HeLa, and the like.

In a preferred embodiment, the invention is directed to methods foradministering peptides of the invention for inhibiting membrane fusioninduced by a paramyxovirus. Membrane fusion is the process whereby thelipid envelope of the virus fuses with the plasma membrane of the hostcell. In vivo this is an essential step in infection. In vitro, theprocess is referred to as syncytial cell formation whereby the plasmamembranes of a plurality of cells combine to form one or more syncytia.

The instant pharmaceutical compositions can also be administered orallyor non-orally in the form of, for example, granules, powders, tablets,capsules, syrup, suppositories, injections, emulsions, elixir,suspensions or solutions, by mixing these effective components,individually or simultaneously, with pharmaceutically acceptablecarriers, excipients, binders, diluents or the like. In the case offormulating the effective components individually, while thusindividually formulated agents can be administered in the form of theirmixture prepared by using, for example, a diluent when administered, theindividually formulated agents can also be administered separately orsimultaneously or with time intervals to the one and same subject.

The instant pharmaceutical compositions of the present invention can beformulated in accordance with conventional procedures. In the presentspecification, “non-orally” include subcutaneous injection, intravenousinjection, intramuscular injections, intraperitoneal injection orinstillation. Injectable preparations, for example, sterile injectableaqueous suspensions or oil suspensions can be prepared by knownprocedure in the fields concerned, using a suitable dispersant orwetting agent and suspending agent. The sterile injections may be in thestate of, for example, a solution or a suspension, which is preparedwith a non-toxic diluent administrable non-orally, e.g. an aqueoussolution, or with a solvent employable for sterile injection. Examplesof usable vehicles or acceptable solvents include water, Ringer'ssolution and an isotonic aqueous saline solution. Further, a sterilenon-volatile oil can usually be employed as solvent or suspending agent.

Any non-volatile oil and a fatty acid can be used for this purpose,which includes natural or synthetic or semi-synthetic fatty acid oil orfatty acid, and natural or synthetic or semi-synthetic mono- or di- ortri-glycerides.

Rectal suppositories can be prepared by mixing the drug with a suitablenon-irritable vehicle, for example, cocoa butter and polyethyleneglycol, which is in the solid state at ordinary temperatures, in theliquid state at temperatures in intestinal tubes and melts in rectum torelease the drug.

As a solid formulation for oral administration, the instant inventioncontemplates, for example, powders, granules, tablets, pills andcapsules. The active component compounds can be mixed with at least oneadditive, for example, sucrose, lactose, cellulose sugar, mannitol,maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins,tragacanth gum, gum arabic, gelatins, collagens, casein, albumin,synthetic or semi-synthetic polymers or glycerides. These formulationscan contain, as in conventional cases, further additives, for example,an inactive diluent, a lubricant such as magnesium stearate, apreservative such as paraben or sorbic acid, an anti-oxidant such asascorbic acid, α-tocopherol or cysteine, a disintegrator, a binder, athickening agent, a buffer, a sweetener, a flavoring agent and aperfuming agent. Tablets and pills can further be prepared with entericcoating. Examples of liquid preparations for oral administration includepharmaceutically acceptable emulsions, syrups, elixirs, suspensions andsolutions, which may contain an inactive diluent, for example, water,which is conventionally employed in the field concerned.

The pharmaceutical composition of this invention may be used as amedicine for animals, such as mammals (e.g. human being, horse, cattle,sheep, dog, rabbit, mouse, etc.), preferably humans.

In a preferred embodiment, the instant invention provides a method fortreating infection with a virus, preferably paramyxoviruses, comprisingadministering an effective amount of a composition of the preferredembodiment. In another preferred embodiment, the invention provides amethod for inhibiting fusion between a membrane of a paramyxovirus and aplasma membrane of a cell comprising administering a compositionaccording to the instant invention. These methods include thealleviation of symptoms associated with viral infection.

The effective amount is that amount which effectively inhibits fusionbetween a membrane of a paramyxovirus and a plasma membrane of a cell,or that which alleviates the symptoms of paramyxovirus infection. Theeffective amount is dependent on the age, body weight, general healthconditions, sex, diet, dose interval, administration routes, excretionrate, combinations of drugs and conditions of the diseases treated,while taking these and other necessary factors into consideration.

Biologically active fragments are those which can inhibit fusion betweena membrane of a paramyxovirus and a plasma membrane of a cell. This maybe easily determined by a cell fusion assy. A cell fusion assay may beutilized to test the peptides' ability to inhibit viral-induced syncytiaformation in vitro. Such an assay is illustrated in the examples below,and may comprise culturing uninfected cells in the presence of cellschronically infected with a syncytial-inducing virus and a peptide to beassayed. For each peptide, a range of peptide concentrations may betested. This range should include a control culture wherein no peptidehas been added. Standard conditions for culturing, well known to thoseof ordinary skill in the art, are used. After incubation for anappropriate period the culture is examined for the presence ofmultinucleated giant cells, which are indicative of cell fusion andsyncytia formation.

Another embodiment of the invention is directed to antibodiesspecifically reactive to isolated peptides of the invention. Antibodiesmay be monoclonal or polyclonal, as desired, and may be of any isotypesuch as IgA, IgD, IgE, IgG, or IgM. Antibodies may be useful fortreatment by interfering with interaction between Henipavirus and a celland thereby preventing infection. Also, antibodies may be useful as adiagnostic for possible or suspected Henipavirus infection by detectingthe presence of F glycoprotein in a sample obtained from the patient.Alternatively, diagnostic kits may comprise peptides of the inventionand be used to detect circulating antibodies.

In another preferred embodiment, the instant invention also providesaptamers of peptides of the instant invention and methods for inhibitingfusion between a membrane of a paramyxovirus and a plasma membrane withthe instant aptamers. The instant aptamers are peptide or nucleic acidmolecules that are capable of binding to a protein or other molecule, ormimic the three dimensional structure of the active portion of thepeptides of the invention, and thereby disturb the protein or othermolecule function. The instant aptamers are based on heptad portion of aHenipavirus F protein and functions to inhibit fusion of a membrane of aparamyxovirus and a plasma membrane of a cell. Aptamers may be preparedby any known method, including synthetic, recombinant, and purificationmethods (Nature 1996 Apr. 11; 380(6574):548-50).

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

The following examples are offered to illustrate embodiments of theinvention, but are not to be viewed as limiting the scope of theinvention.

EXAMPLES

Nipah virus (NiV) and Hendra virus (HeV) are viruses that resulted inpreviously unrecognized fatal diseases in animals and humans. Nipahvirus and Hendra virus are closely related members of a new genus,Henipavirus within the family Paramyxoviridae, a diverse group of large,enveloped, negative-sense single stranded RNA viruses, and include avariety of important human and animal pathogens. The recent emergenceand discovery of these two viruses appears to have been the result ofexposure of new hosts precipitated by certain economical, environmental,or behavioral changes. Hendra virus was identified first, from cases ofsevere respiratory disease that fatally affected both horses and man.Subsequent to that appearance, an outbreak of severe febrileencephalitis associated with human deaths occurred in Malaysia. Laterstudies identified a Hendra-like virus, now known as Nipah virus, as theetiologic agent of that episode. These viruses are unusual among theparamyxoviruses in their abilities to infect and cause potentially fataldisease in a number of host species, including humans, and are zoonoticBiological Safety Level-4 (BSL4) agents. Presently, the cat appears tobe the ideal small animal model capable of reproducing the pathologyseen in Nipah virus infected humans. Nipah and Hendra virus possessseveral biological features which make them highly adaptable for theiruse as biowarfare agents and are select agents of biodefense importance.The development of effective therapies or vaccines for these agents iscritical.

The instant polypeptides are derived from the carboxyl (C)-terminalc-helical heptad repeat region of the NiV and HeV fusion glycoprotein(F), and block the virus-mediated fusion step by interfering with theformation of a trimer-of-hairpins structure in the fusion glycoprotein,which has been similarly demonstrated in a number of viral systems thatpossess a pH-independent mode of membrane fusion. In the absence of anypassive or active immunization procedures or products to NiV and HeV, aswell as no available anti-viral drugs for paramyxoviruses in general;the instant polypeptides developed here may well represent an effectivetherapy for NiV and HeV infection resulting from their use as abiological weapon, a natural outbreak, or a BSL4 laboratory accident.

The instant polypeptides are an effective therapeutic treatment for bothNiV and HeV infection in animals and humans, such that acuteencephalitic disease or death, may be prevented. The protectivemechanism of the instant polypeptides will be to afford thevirus-infected host an opportunity to mount a sterilizing immuneresponse.

Example 1

The location of HeV F glycoprotein heptad repeats is shown in FIG. 1. Adiagram of the HeV F glycoprotein depicting important structural andfunctional elements is depicted in FIG. 1A. Amino acid sequence of theC-terminal heptad repeat of HeV F (SEQ ID NO 1) is depicted in FIG. 1B.A helical wheel representation of the N- and C-terminal heptad repeatsof HeV F is depicted in FIG. 1C. The bold-faced points on the helicalwheel indicate important residue locations on the helix structure of theF protein of SVS that mediate protein-protein interactions (Joshi,Dutch, and Lamb, 1998). The point “a” of one N-terminal heptad isthought to interact with point “d” of another SVS F N-terminal heptad inan antiparallel orientation. Point “e” of the N-terminal heptad isthought to interact with point “a” of the C-terminal heptad, and point“g” of the N-terminal heptad is believed to interact with point “d” ofthe C-terminal heptad. In all, it is thought that three N-terminalheptad repeats and three C-terminal heptad repeats of three SV5 Fproteins mediate the necessary protein-protein interactions thatstabilize the fusogenic SV5 F trimer formation. Enlarged underlinedamino acids represent HeV F residues that are identical to those foundin the N- and C-terminal heptad repeats of SV5, enlarged but notunderlined amino acids are hydrophobic conservative substitutions in HeVF as compared to SV5 F.

Example 2 Specific Heptad Peptide Inhibition of NiV and HeV-mediatedMembrane Fusion and Virus Infection

To further assess the specificity and utility of the fusion system, wayswere sought to specifically inhibit the cell-fusion process. Sequenceswhich formed a trimer-of-hairpins structure whose oligomeric coiled-coilformation is mediated by the 2 α-helical heptad repeat domains of thefusion protein were investigated first. Peptides derived from either ofthe α-helical heptad repeat regions of enveloped viral fusion proteinsare potentially potent inhibitors of the fusion process for a number ofviruses. There are 2 putative α-helical domains in the HeV Fglycoprotein. One HeV F heptad domain is proximal to the fusion peptideof F₁ (N-terminal heptad repeat), and the other is very close to thepredicted transmembrane domain of F₁ (C-terminal heptad repeat). Helicalwheel analysis of HeV F revealed a high degree of sequence homology ofimportant functional residues with the heptad repeats of SV5 F, and bothHeV and NiV have two putative heptad repeat domains in F. To determineif these domains played important roles in NiV and HeV-mediated fusion,a 42 amino acid peptide analogous to the NiV F C-terminus heptad repeatwas synthesized (NiV FC1) and tested for its ability to interfere withNiV-mediated fusion. Since there were three amino acid differenceswithin the C-terminus heptad repeat of HeV and NiV, a second peptidecorresponding to the HeV F C-terminus heptad repeat was also synthesized(HeV FC2). A scrambled version of HeV FC2 was synthesized and used as anegative control (Table 1).

Specificity of NiV and HeV-mediated fusion was determined. Briefly, HeLacells were infected with vaccinia recombinants encoding NiV or HeV F andG. Human U373 cells were infected with the E. coli LacZ-encodingreporter vaccinia virus vCB21R (target cells). Peptides were diluted andadded to the glycoprotein-expressing cells. Cell populations were mixedand cell fusion was measured. Each peptide and serum concentration wasperformed in duplicate. Shown in FIGS. 2A and 2B are the resultsobtained in the presence of these peptides for both HeV and NiV-mediatedfusion. TABLE 1 Synthetic peptides corresponding to HeV and NiV FPeptide Sequence Target HeV-FC2 PPVYTDKVDISSQISSMNQSL C-terminal SEQ IDNO 1 QQSKDYIKEAQKILDTVNPSL Heptad repeat NiV-FCl PPVFTDKVDISSQISSMNQSLC-terminal SEQ ID NO 2 QQSKDYIKEAQRLLDTVNPSL Heptad repeat ScHeV FC2KPYTQSDVSMISLQSQKSINS Scrambled LPSQIKDYVQKTVILAEDNP control

HeV FC2 and NiV FC1 could inhibit both HeV and NiV-mediated fusion in adose dependent manner and was completely inhibitory in the nM range,with IC₅₀ values between 5.2 and 5.8 nM, respectively. The ScHeV FC2 hadno inhibitory effect on HeV or NiV-fusion. These data indicate that bothviruses have a similar mechanism of fusion, that is likely comparable tothat proposed for other viral systems where a trimer of hairpins hasbeen hypothesized to form. There were no significant differences in theability of HeV FC2 and NiV FC1 to neutralize either HeV or NiV-mediatedfusion. The conservative Y450F or K479R substitutions of NiV F did notaffect the ability of NiV FC1 to inhibit HeV fusion or the ability ofHeV FC2 to inhibit NiV fusion. These results are further supported byhelical wheel analysis which revealed that none of these amino acidsfall in the proposed functional points of the putative C-terminalα-helix of HeV and NiV F thought to be involved in protein-proteininteractions leading to the formation of the trimer-of-hairpin fusogenicconformation. Thus, a single heptad peptide could be used to inhibit thefusion of either virus.

In addition, the HeV-FC1 peptide has been evaluated as an inhibitor oflive HeV infection under BSL4 containment. Shown in Table 2 and 3, areresults obtained with the HeV-FC1 peptide inhibition of live HeV and NiVinfection of Vero cells. In these experiments, 44 and 49 foci formingunits (infectious units) of HeV and NiV respectively were mixed for 30minutes with peptide at the concentrations given. Virus-peptide mixtureswere adsorbed to monolayers of Vero cells in chamber slides for 30minutes at room temperature. Virus-peptide mixtures were removed andmedium containing the same concentration of peptide was added. After 24hours incubation at 37° C. monolayers were fixed with methanol andremoved from BSL4 containment. The number of foci of infection wasdetermined by immunofluorescence using mono-specific rabbit anti-HeV Pprotein antiserum. Each focus of infection was a syncytium. The numberof nuclei in each syncytium was readily determined except in the control(no peptide) where a proportion of syncytia contained over 60 nuclei persyncytium and in which the number of nuclei could not be accuratelydetermined. At a concentration of about 3.5 μM the number of foci isapproximately 50% that of the control value, a concentration quitecomparable to other paramyxovirus systems using heptad peptides withlive virus in a cell culture model. TABLE 2 Inhibition of HeV Infectionby Heptad Peptide FC1 Peptide #Virus- # Nuclei per % Inhibition ofconcentration infected foci focus/syncytium focus formation 112 μM 2.31.00 94.78 56 μM 5.7 1.18 87.14 14 μM 20.0 7.80 54.55 3.5 μM 19.7 9.5055.23 875 nM 41.0 13.71 6.80 219 nM 40.5 17.80 7.95 55 nM 43.0 17.202.27 none 44.0 >44 0.0

TABLE 3 Inhibition of NiV Infection by Heptad Peptide FC1 Peptide#Virus- # Nuclei per % Inhibition of concentration infected focifocus/syncytium focus formation 112 μM 4 0.25 91.8 56 μM 4 0.75 91.8 14μM 18 7.5 63.26 3.5 μM 19 8.9 61.2 875 nM 37 15.6 24.5 219 nM 38 17.822.4 55 nM 56 28.63 −14.28 None 49 >55 0.0There also was a significant reduction in the size of the syncytium, asdetermined by the number of nuclei even at the lowest peptideconcentration tested.

Next, peptides were analyzed for their ability to prevent infection byNiV as determined by syncytia formation. Briefly, approximately 80 fociforming units (infectious units) of NiV were mixed for 30 min withpeptides at the concentrations given and virus-peptide mixtures wereadsorbed to monolayers of Vero cells in chamber slides for 30 min at RT.Virus-peptide mixtures were removed and medium containing the sameconcentration of peptide was added. After 24 hours at 37° C., monolayerswere fixed with methanol and the number of foci of infection wasdetermined by immuno-fluorescence using mono-specific anti-HeV Pantiserum.

As shown in FIG. 3, the number of nuclei in NiV-induced syncytia can bereduced to less than 30% of that in control untreated syncytia by bothFC1 and FC2 peptides at 32 nM concentration (FIG. 2). This valueapproaches the IC₅₀ value for peptides FC1 and FC2 in the in vitrofusion assay. These data indicate that these heptad peptides mayrepresent a therapeutic avenue for both HeV and NiV infections in humansto prevent lethal disease outcomes, such as preventing viralCNS-transition and acute encephalitic disease, and affording theinfected host the an opportunity to mount a sterilizing immune response.The humoral and cellular immune responses to these viruses, like measlesvirus and mumps virus, which also cause systemic disease, is vigorous,potent, and capable of neutralizing low passage virus.

Taken together, in the absence of any passive or active immunizationprocedures or products to Nipah and Hendra virus, as well as noavailable anti-viral drugs for paramyxoviruses in general; the heptadpeptides of the instant invention may well represent an effectivetherapy for Nipah and Hendra virus infection resulting from their use asa biological weapon, a natural outbreak, or a BSL4 laboratory accident.The instant heptad peptides are an effective therapeutic treatment forboth Nipah and Hendra virus infection in animals, such as mammals andpreferably humans, such that acute encephalitic disease or death, may beprevented. The instant heptad peptides afford the virus-infected host anopportunity to mount a sterilizing immune response.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein for anyreason, including all U.S. and foreign patents and patent applications,are specifically and entirely incorporated by reference. It is intendedthat the specification and examples be considered exemplary only, withthe true scope and spirit of the invention indicated by the followingclaims.

REFERENCES

-   ALKHATIB, G., BRODER, C. C., and BERGER, E. A. (1996). Cell    type-specific fusion cofactors determine human immunodeficiency    virus type 1 tropism for T-cell lines versus primary macrophages. J    Virol 70(8), 5487-94.-   ANONYMOUS. (1999). From the Centers for Disease Control and    Prevention. Outbreak of Hendra-like virus—Malaysia and Singapore,    1998-1999. Jama 281(19), 1787-8.-   BAGAI, S., and LAMB, R. A. (1995). Quantitative measurement of    paramyxovirus fusion: differences in requirements of glycoproteins    between simian virus 5 and human parainfluenza virus 3 or Newcastle    disease virus. J Virol 69(11), 6712-9.-   BERGER, E. A., NUSSBAUM, O., and BRODER, C. C. (1995). HIV envelope    glycoprotein/CD4 interactions: studies using recombinant vaccinia    virus vectors. In “HIV: a Practical Approach, Volume II” (J. Karn,    Ed.), Vol. 2, pp. 123-145. Oxford University Press, Cambridge.-   BRODER, C. C., and BERGER, E. A. (1995). Fusogenic selectivity of    the envelope glycoprotein is a major determinant of human    immunodeficiency virus type 1 tropism for CD4+ T-cell lines vs.    primary macrophages. Proc Natl Acad Sci USA 92(19), 9004-8.-   BRODER, C. C., DIMITROV, D. S., BLUMENTHAL, R., and BERGER, E. A.    (1993). The block to HIV-1 envelope glycoprotein-mediated membrane    fusion in animal cells expressing human CD4 can be overcome by a    human cell component(s). Virology 193(1), 483-91.-   BRODER, C. C., and EARL, P. L. (1999). Recombinant vaccinia viruses.    Design, generation, and isolation. Mol Biotechnol 13(3), 223-45.-   BRODER, C. C., NUSSBAUM, O., GUTHEIL, W. G., BACHOVCHIN, W. W., and    BERGER, E. A. (1994). CD26 antigen and HIV fusion? Science 264,    1156-1159.-   CARROLL, M. W., and Moss, B. (1995). E. coli beta-glucuronidase    (GUS) as a marker for recombinant vaccinia viruses. Biotechniques    19(3), 352-4, 356.-   CHAN, D. C., and KIM, P. S. (1998). HIV entry and its inhibition.    Cell 93(5), 681-4.-   CHUA, K. B., BELLINI, W. J., ROTA, P. A., HARCOURT, B. H., TAMIN,    A., LAM, S. K., KSIAZEK, T. G., ROLLIN, P. E., ZAKI, S. R., SHIEH,    W., GOLDSMITH, C. S., GUBLER, D. J., ROEHRIG, J. T., EATON, B.,    GOULD, A. R., OLSON, J., FIELD, H., DANIELS, P., LING, A. E.,    PETERS, C. J., ANDERSON, L. J., and MAHY, B. W. (2000). Nipah virus:    a recently emergent deadly paramyxovirus. Science 288(5470), 1432-5.-   CHUA, K. B., GOH, K. J., WONG, K. T., KAMARULZAMAN, A., TAN, P. S.,    KSIAZEK, T. G., ZAKI, S. R., PAUL, G., LAM, S. K, and TAN, C. T.    (1999). Fatal encephalitis due to Nipah virus among pig-farmers in    Malaysia. Lancet 354(9186), 1257-9.-   CHUNG, M., KIZHATIL, K., ALBRITTON, L. M., and GAULTON, G. N.    (1999). Induction of syncytia by neuropathogenic murine leukemia    viruses depends on receptor density, host cell determinants, and the    intrinsic fusion potential of envelope protein. J Virol 73(11),    9377-85.-   DANIELS, P., KSIAZEK, T., and EATON, B. T. (2001). Laboratory    diagnosis of Nipahand Hendra virus infections. Microbes Infect 3(4),    289-95.-   DOMS, R. W., LAMB, R., ROSE, J. K., and HELENIUS, A. (1993). Folding    and assembly of viral membrane proteins. Virology 193, 545-562.-   EATON, B. T. (2001). Introduction to Current focus on Hendra and    Nipah viruses. Microbes Infect 3(4), 277-8.-   FENG, Y., BRODER, C. C., KENNEDY, P. E., and BERGER, E. A. (1996).    HIV-1 entry cofactor: functional cDNA cloning of a    seven-transmembrane, G protein-coupled receptor. Science 272(5263),    872-7.-   FIELD, H., YOUNG, P., YOB, J. M., MILLS, J., HALL, L., and    MACKENZIE, J. (2001). The natural history of Hendra and Nipah    viruses. Microbes Infect 3(4), 307-14.-   FUERST, T. R., NILES, E. G., STUDIER, F. W., and Moss, B. (1986).    Eukaryotic transient-expression system based on recombinant vaccinia    virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl.    Acad. Sci. USA 83, 8122-8126.-   GOH, K. J., TAN, C. T., CHEW, N. K., TAN, P. S., KAMARULZAMAN, A.,    SARJI, S. A., WONG, K T., ABDULLAH, B. J., CHUA, K B., and    LAM, S. K. (2000). Clinical features of Nipah virus encephalitis    among pig farmers in Malaysia. N Engl J Med 342(17),1229-35.-   GOULD, A. R. (1996). Comparison of the deduced matrix and fusion    protein sequences of equine morbillivirus with cognate genes of the    Paramyxoviridae. Virus Res 43(l), 17-31.-   HALPIN, K, YOUNG, P. L., FIELD, H., and MACKENZIE, J. S. (1999).    Newly discovered viruses of flying foxes. Vet Microbiol 68(1-2),    83-7.-   HALPIN, K, YOUNG, P. L., FIELD, H. E., and MACKENZIE, J. S. (2000).    Isolation of Hendra virus from pteropid bats: a natural reservoir of    Hendra virus. J Gen Virol 81(Pt 8), 1927-1932.-   HERNANDEZ, L. D., HOFFMAN, L. R., WOLFSBERG, T. G., and WHITE, J. M.    (1996). Virus-cell and cell-cell fusion. Annu Rev Cell Dev Biol 12,    627-61.-   HOOPER, P., ZAKI, S., DANIELS, P., and MIDDLETON, D. (2001).    Comparative pathology of the diseases caused by Hendra and Nipah    viruses. Microbes Infect 3(4), 315-22.-   HUGHSON, F. M. (1997). Enveloped viruses: a common mode of membrane    fusion? Curr Biol 7(9), R565-9.-   HUNTER, E. (1997). Viral entry and receptors. In “Retroviruses”    (S. H. Coffin, S. H. Hughes, and H. E. Varmus, Eds.), pp. 71-119.    Cold Spring Harbor Laboratory Press, New York.-   JIANG, S., LIN, K., STRICK, N., and NEURATH, A. R. (1993). HIV-1    inhibition by a peptide. Nature 365, 113.-   JOSHI, S. B., DUTCH, R. E., and LAMB, R. A. (1998). A core trimer of    the paramyxovirus fusion protein: parallels to influenza virus    hemagglutinin and HIV-1 gp41. Virology 248(1), 20-34.-   KLENK, H. D., and GARTEN, W. (1994). Host cell proteases controlling    virus pathogenicity. Trends Microbiol 2(2), 39-43.-   KRUEGER, D. K., KELLY, S. M., LEWICKI, D. N., RUFFOLO, R., and    GALLAGHER, T. M. (2001). Variations in disparate regions of the    murine coronavirus spike protein impact the initiation of membrane    fusion. J Virol 75(6), 2792-802.-   LAMB, R. A. (1993). Paramyxovirus fusion: A hypothesis for changes.    Virology 197, 1-11.-   LAMB, R. A., JOSHI, S. B., and DUTCH, R. E. (1999). The    paramyxovirus fusion protein forms an extremely stable core trimer:    structural parallels to influenza virus haemagglutinin and HIV-1    gp41. Mol Membr Biol 16(1), 11-9.-   LAMB, R. A., and KOLAKOFSKY, D. (1996). Paramyxoviridae: The viruses    and their replication. 3 ed. In “Virology” (B. N. Fields, D. M.    Knipe, and P. M. Howley, Eds.), pp. 1177-1204. Raven Press, New    York.-   LEE, K. E., UMAPATHI, T., TAN, C. B., TJIA, H. T., CHUA, T. S.,    OH, H. M., FOCK, K. M., KURUP, A., DAS, A., TAN, A. K., and    LEE, W. L. (1999). The neurological manifestations of Nipah virus    encephalitis, a novel paramyxovirus. Ann Neurol 46(3), 428-32.-   LIM, C. C., SITOH, Y. Y., HUI, F., LEE, K. E., ANG, B. S., LIM, E.,    LIM, W. E., OH, H. M., TAMBYAH, P. A., WONG, J. S., TAN, C. B., and    CHEE, T. S. (2000). Nipah viral encephalitis or Japanese    encephalitis? MR findings in a new zoonotic disease. AJNR Am J    Neuroradiol 21(3), 455-61.-   MICHALSKI, W. P., CRAMERI, G., WANG, L., SHIELL, B. J., and    EATON, B. (2000). The cleavage activation and sites of glycosylation    in the fusion protein of Hendra virus. Virus Res 69(2), 83-93.-   MUNOZ-BARROSO, I., DURELL, S., SAKAGUCHI, K., APPELLA, E., and    BLUMENTHAL, R. (1998). Dilation of the human immunodeficiency    virus-1 envelope glycoprotein fusion pore revealed by the inhibitory    action of a synthetic peptide from gp41. J Cell Biol 140(2),315-23.-   MURRAY, K., EATON, B., HOOPER, P., WANG, L., WILLIAMSON, M., and    YOUNG, P. (1998). Flying Foxes, Horses, and Humans: a Zoonosis    Caused be a New Member of the Paramyxoviridae. In “Emerging    Infections” (W. M. Scheld, D. Armstrong, and J. M. Hughes, Eds.),    pp. 43-58. ASM Press, Washington, D.C.-   MURRAY, K., SELLECK, P., HOOPER, P., HYATT, A., GOULD, A., GLEESON,    L., WESTBURY, H., HILEY, L., SELVEY, L., RODWELL, B., and ET AL.    (1995). A morbillivirus that caused fatal disease in horses and    humans. Science 268(5207), 94-7.-   NANICHE, D., VARIOR-KRISHNAN, G., CERVONI, F., WILD, T. F., ROSSI,    B., RABOURDIN-COMBE, C., and GERLIER, D. (1993). Human membrane    cofactor protein (CD46) acts as a cellular receptor for measles    virus. J Virol 67(10), 6025-32.-   NUSSBAUM, O., BRODER, C. C., and BERGER, E. A. (1994). Fusogenic    mechanisms of enveloped-virus glycoproteins analyzed by a novel    recombinant vaccinia virus-based assay quantitating cell    fusion-dependent reporter gene activation. J Virol 68(9), 5411-22.-   NUSSBAUM, O., BRODER, C. C., Moss, B., STERN, L. B., ROZENBLATT, S.,    and BERGER, E. A. (1995). Functional and structural interactions    between measles virus hemagglutinin and CD46. J Virol 69(6), 3341-9.-   O'SULLIVAN, J. D., ALLWORTH, A. M., PATERSON, D. L., SNOW, T. M.,    BOOTS, R., GLEESON, L. J., GOULD, A. R., HYATT, A. D., and    BRADFIELD, J. (1997). Fatal encephalitis due to novel paramyxovirus    transmitted from horses. Lancet 349(9045), 93-5.-   PASTEY, M. K, and SAMAL, S. K. (1997). Analysis of bovine    respiratory syncytial virus envelope glycoproteins in cell fusion. J    Gen Virol 78(Pt 8), 1885-9.-   PATERSON, R. G., JOHNSON, M. L., and LAMB, R. A. (1997).    Paramyxovirus fusion (F) protein and hemagglutinin-neuraminidase    (HN) protein interactions: intracellular retention of F and HN does    not affect transport of the homotypic HN or F protein. Virology    237(1), 1-9.-   RIMSKY, L. T., SHUGARS, D. C., and MATTHEWS, T. J. (1998).    Determinants of human immunodeficiency virus type I resistance to    gp41-derived inhibitory peptides. J Virol 72(2), 986-93.-   ROOT, M. J., KAY, M. S., and KIM, P. S. (2001). Protein design of an    HIV-1 entry inhibitor. Science 291(5505), 884-8.-   SANTORO, F., KENNEDY, P. E., LOCATELLI, G., MALNATI, M. S.,    BERGER, E. A., and LUSSO, P. (1999). CD46 is a cellular receptor for    human herpesvirus 6. Cell 99(7), 817-27.-   SCHEID, A., and CHOPPIN, P. W. (1974). Identification of biological    activities of paramyxovirus glycoproteins. Activation of cell    fusion, hemolysis, and infectivity of proteolytic cleavage of an    inactive precursor protein of Sendai virus. Virology 57(2), 475-90.-   SINGH, M., BERGER, B., and KIM, P. S. (1999). LeamCoil-VMF:    computational evidence for coiled-coil-like motifs in many viral    membrane-fusion proteins. J Mol Biol 290(5), 1031-41.-   SKEHEL, J. J., and WILEY, D. C. (1998). Coiled coils in both    intracellular vesicle and viral membrane fusion. Cell 95(7), 871-4.-   TAKIKAWA, S., ISHII, K., AIZAKI, H., SUZUKI, T., ASAKURA, H.,    MATSUURA, Y., and MIYAMURA, T. (2000). Cell fusion activity of    hepatitis C virus envelope proteins. J Virol 74(11), 5066-74.-   WANG, L., HARCOURT, B. H., YU, M., TAMIN, A., ROTA, P. A.,    BELLINI, W. J., and EATON, B. T. (2001). Molecular biology of Hendra    and Nipah viruses. Microbes Infect 3(4), 279-87.-   WANG, L. F., and EATON, B. T. (2001). Henipavirus (Paramyxoviridae).    In “The Springer Index of Viruses” (C. A. Tidona, and G. Darai,    Eds.), pp. In Press. Springer-Verlag, Berlin/Heidelberg.-   WANG, L. F., MICHALSKI, W. P., YU, M., PRITCHARD, L. I., CRAMERI,    G., SHIELL, B., and EATON, B. T. (1998). A novel P/V/C gene in a new    member of the Paramyxoviridae family, which causes lethal infection    in humans, horses, and other animals. J Virol 72(2), 1482-90.-   WANG, L. F., YU, M., HANSSON, E., PRITCHARD, L. I., SHIELL, B.,    MICHALSKI, W. P., and EATON, B. T. (2000). The exceptionally large    genome of Hendra virus: support for creation of a new genus within    the family Paramyxoviridae. J Virol 74(21), 9972-9.-   WEISSENHORN, W., DESSEN, A., CALDER, L. J., HARRISON, S. C.,    SKEHEL, J. J., and WILEY, D. C. (1999). Structural basis for    membrane fusion by enveloped viruses. Mol Membr Biol 16(1),3-9.-   WESTBURY, H. A., HOOPER, P. T., SELLECK, P. W., and MURRAY, P. K.    (1995). Equine morbillivirus pneumonia: susceptibility of laboratory    animals to the virus. Aust Vet J 72(7), 278-9.-   WILD, C. T., SHUGARS, D. C., GREENWELL, T. K., MCDANAL, C. B., and    MATTHEWS, T. J. (1994). Peptides corresponding to a predictive    alpha-helical domain of human immunodeficiency virus type I gp4l are    potent inhibitors of virus infection. Proc Natl Acad Sci U S A    91(21), 9770-4.-   WILD, T. F., and BUCKLAND, R. (1997). Inhibition of measles virus    infection and fusion with peptides corresponding to the leucine    zipper region of the fusion protein. J Gen Virol 78(Pt 1), 107-11.-   YAO, Q., HU, X., and COMPANS, R. W. (1997). Association of the    parainfluenza virus fusion and hemagglutinin-neuraminidase    glycoproteins on cell surfaces. J Virol 71(1), 650-6.-   YOUNG, P. L., HALPIN, K., SELLECK, P. W., FIELD, H., GRAVEL, J. L.,    KELLY, M. A., and MACKENZIE, J. S. (1996). Serologic evidence for    the presence in Pteropus bats of a paramyxovirus related to equine    morbillivirus. Emerg Infect Dis 2(3), 239-40.-   YU, M., HANSSON, E., LANGEDUK, J. P., EATON, B. T., and WANG, L. F.    (1998a). The attachment protein of Hendra virus has high structural    similarity but limited primary sequence homology compared with    viruses in the genus Paramyxovirus. Virology 251(2), 227-33.-   YU, M., HANSSON, E., SHIELL, B., MICHALSKI, W., EATON, B. T., and    WANG, L. F. (1998b). Sequence analysis of the Hendra virus    nucleoprotein gene: comparison with other members of the subfamily    Paramyxovirinae. J Gen Virol 79(Pt 7), 1775-80.

1. An isolated polypeptide which is a heptad portion of a Henipavirus Fprotein that function to inhibit fusion of a membrane of a paramyxovirusand a plasma membrane of a cell.
 2. The polypeptide of claim 1 whereinsaid polypeptide comprises a biologically active fragment of a heptadportion of a Henipavirus F protein that functions to inhibit fusion of amembrane of a paramyxovirus and a plasma membrane of a cell.
 3. Thepolypeptide of claim 1 wherein said polypeptide comprises a deletion,substitutional, or insertional variant of a heptad portion of aHenipavirus F protein that functions to inhibit fusion of a membrane ofa paramyxovirus and a plasma membrane of a cell.
 4. The polypeptide ofclaim 1 wherein said polypeptide is recombinant.
 5. The peptide of claim1 wherein the polypeptide is derived from HeV or NiV.
 6. A polypeptidethat comprises the polypeptide sequence of SEQ ID NO 1 or SEQ ID NO 2.7. The polypeptide of claim 6 wherein said polypeptide comprises abiologically active fragment of SEQ ID NO 1 or SEQ ID NO
 2. 8. Thepolypeptide of claim 6 wherein said polypeptide comprises a deletion,substitutional, or insertional variant of SEQ ID NO 1 or SEQ ID NO
 2. 9.The polypeptide of claim 6 wherein said polypeptide is recombinant. 10.The peptide of claim 6 wherein the polypeptide is derived from HeV orNiV.
 11. A pharmaceutical composition comprising an effective amount ofa polypeptide sequence of SEQ ID NO 1 or SEQ ID NO 2 and apharmaceutically acceptable carrier.
 12. The composition of claim 11comprising a biologically active fragment of SEQ ID NO 1 or SEQ ID NO 2.13. The composition of claim 11 comprising a deletion, substitutional,or insertional variant of SEQ ID NO 1 or SEQ ID NO
 2. 14. Thecomposition of claim 11 wherein the composition is a therapeutic orpost-exposure prophylactic.
 15. The composition of claim 11 wherein thecomposition is a vaccine.
 16. A method for inhibiting fusion between amembrane of a paramyxovirus and a plasma membrane of a cell comprisingadministering a composition according to claim
 11. 17. The method ofclaim 16 wherein said paramyxovirus is of the genus Henipavirus.
 18. Themethod of claim 16 wherein said paramyxovirus is of the subfamilyParamyxovirina.
 19. The method of claim 16 wherein said paramyxovirus isHeB or NiV.
 20. An isolated polynucleotide sequence encoding apolypeptide that inhibits fusion between a membrane of a paramyxovirusand a plasma membrane of a cell, wherein said polynucleotide sequence isselected from the group consisting of: a DNA sequence encoding apolypeptide of SEQ ID NO 1; and a DNA sequence capable of hybridizingunder high stringency conditions to the complement of a DNA sequenceencoding a polypeptide of SEQ ID NO
 1. 21. A vector comprising apolynucleotide sequence of claim
 20. 22. A cell comprising apolynucleotide sequence of claim
 20. 23. A method for treating infectionwith a virus, comprising administering the composition of claim
 11. 24.The method of claim 23 wherein said virus is a paramyxovirus.
 25. Themethod of claim 23 wherein said paramyxovirus is of the genusHenipavirus.
 26. The method of claim 23 wherein said virus is HeV orNiV.
 27. An aptamer of a heptad portion of a Henipavirus F protein thatfunctions to inhibit fusion of a membrane of a paramyxovirus and aplasma membrane of a cell.
 28. An aptamer of a polypeptide sequence ofSEQ ID NO 1 or SEQ ID NO
 2. 29. The biologically active fragment of anaptamer of claim
 27. 30. A method for inhibiting fusion between amembrane of a paramyxovirus and a plasma membrane of a cell comprisingadministering an effective amount of an aptamer of claim 27.